This is a competitive renewal of a grant proposal that during the initial funding period defined the importance of RANKL in the osteolysis that occurs around total joint replacements. There are currently more than 400,000 arthroplasties performed annually in the U.S. to treat this condition. Since up to 20% of arthroplasties require revision surgery due to aseptic loosening, this complication of arthritic treatment constitutes a major source of the morbidity, and represents billions of dollars in health care costs. During the last funding period, our work defined the interfascial membrane synovial fibroblast as a major source of RANKL. Preliminary data show that RANKL expression is dependent upon the stimulation of COX-2 and PGE2 production by Ti particles. Moreover, our findings suggest that the EP4 receptor is involved in the induction of RANKL in synovial fibroblasts. Aim 1 defines the signaling mechanisms through which particulate debris stimulate COX-2 in synovial fibroblasts and initiates intracellular signals leading to the expression of RANKL. Our hypothesis is that the transcription factor NFicB initiates the synovial fibroblast response to particles and is necessary for COX-2 induction. Aim 2 assesses in vitro responses to particles in synovial fibroblast from mice lacking the various EP receptors and examines the ability of these cells to stimulate osteoclastogenesis in co-cultures with osteoclast precursors. We anticipate that the EP4 receptor will be required for RANKL induction and osteoclast formation by Ti treated synovial fibroblasts. Finally, Aim 3 uses an in vivo model of calvarial bone loss in mice lacking EP receptor signaling to definitively address signals involved in RANKL induction, osteoclast formation, gene expression, and osteolysis. The role of the synovial fibroblast will be confirmed by conditional deletion of the EP4 receptor in fibroblasts. Thus through a series of highly integrated in vitro and in vivo experiments, the competitive renewal advances the findings of the initial funding period. The use of transgenic models and state of the art methods will characterize the sequential events of NFicB activation, COX-2 and PGES1 expression, PGE2 secretion, EP4 receptor binding, and RANKL expression in SF as a critical event in osteolysis. [unreadable] [unreadable]